Parallel flow cytometer using radiofrequency multiplexing

ABSTRACT

An imaging flow cytometry apparatus and method which allows registering multiple locations across a cell, and/or across multiple flow channels, in parallel using radio-frequency-tagged emission (FIRE) coupled with a parallel optical detection scheme toward increasing analysis throughput. An optical source is modulated by multiple RF frequencies to produce an optical interrogation beam having a spatially distributed beat frequency. This beam is directed to one or more focused streams of cells whose responsive fluorescence, in different frequencies, is registered in parallel by an optical detector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/672,051 filed on Aug. 8, 2017, now U.S. Pat. No. 10,036,699 which isa continuation of U.S. patent application Ser. No. 15/263,419 filed onSep. 13, 2016, now U.S. Pat. No. 9,784,661, which is a 35 U.S.C. §111(a) continuation of PCT international application numberPCT/US2015/021264 filed on Mar. 18, 2015, incorporated herein byreference in its entirety, which claims priority to, and the benefit of,U.S. provisional patent application Ser. No. 61/955,137 filed Mar. 18,2014, incorporated herein by reference in its entirety. Priority isclaimed to each of the foregoing applications.

The above-referenced PCT international application was published as PCTInternational Publication No. WO 2015/143041 on Sep. 24, 2015, whichpublication is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant NumberW81XWH-10-1-0518, awarded by the U.S. Army, Medical Research andMateriel Command. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF COMPUTER PROGRAM APPENDIX

Not Applicable

NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

A portion of the material in this patent document is subject tocopyright protection under the copyright laws of the United States andof other countries. The owner of the copyright rights has no objectionto the facsimile reproduction by anyone of the patent document or thepatent disclosure, as it appears in the United States Patent andTrademark Office publicly available file or records, but otherwisereserves all copyright rights whatsoever. The copyright owner does nothereby waive any of its rights to have this patent document maintainedin secrecy, including without limitation its rights pursuant to 37C.F.R. § 1.14.

BACKGROUND 1. Technological Field

This technical disclosure pertains generally to flow cytometry, and moreparticularly to a parallel flow channel flow cytometer utilizing opticalbeams uniquely modulated for each flow channel.

2. Background Discussion

Flow cytometry is a biotechnology utilized to analyze the physical andchemical characteristics of particles in a fluid. Flow cytometry isutilized in cell counting, cell sorting, biomarker detection and othermicrobiological and medical processes. Cells are suspended in a streamof fluid in a channel(s) which pass by an optical detection apparatus.Various physical and chemical characteristics of the cells are analyzed(multiparametric analysis).

Applications for flow cytometry include diagnostics, clinical, andresearch, including immunology, virology, hematology, and drugdiscovery, and so forth. It will be noted that drug discovery is anextremely expensive and lengthy process, in which high speed cytometryplays a key role. Development costs are in the billions of dollars,spanning over more than a decade. Average costs have been seen over $5billion to develop each drug, which is partially due to the fact thatfor every drug developed successfully, several fail. Perhaps even moreproblematic is that even with steadily increasing discovery anddevelopment costs, the efficiency of finding new drugs is decreasing. Ithas been reported that the number of drugs discovered per billiondollars spent is halving approximately every 9 years. A strong incentivethus exists to improve all aspects of the drug discovery process andelsewhere.

Within the overall drug discovery pipeline, the most common techniquefor discovering lead compounds that eventually become drugs is highthroughput screening (HTS). In HTS, hundreds of thousands (or evenmillions) of compounds are assayed against a disease target. Today, thiscostly and lengthy process is often performed in large-scalelaboratories, often involving automated robotics and instrumentation,alongside high performance computing. Within the HTS field, it is widelyacknowledged that there is a general and pressing need for inexpensivecompound screening assays and tools that quickly yield accurate, highcontent data in order to reduce the cost of drug discovery and thetime-to-market of novel therapeutic agents. Any technique that canprovide even a moderate advance in this area has an enormous potentialto dramatically reduce costs and improve the overall efficiency.

Flow cytometry is also an established research tool used in many areasof cell biology that provides high content single-cell data by using acombination of optical scattering and multi-color fluorescencemeasurements. While not yet widely used in high throughput compoundscreening, the multi-parameter, phenotypic information yielded by flowcytometry offers significant advantages over the conventional approachof using several separate single-parameter, population-averagedmeasurements to determine the effect of a candidate compound on atarget. By measuring many parameters simultaneously from populations ofsingle cells using flow cytometry, complex intra- and inter-cellularinteractions within a cell or cellular population can be more quicklyelucidated than with well-level screens (e.g., luminescence, absorbance,ELISA, NMR, time resolved fluorescence, FRET, and the like). This highcontent, multiparameter data inherently yields deeper insight into theeffect a compound may ultimately have on a patient during clinicaltrials, and may potentially reduce or eliminate the need for furtherdownstream assays in the drug discovery pipeline (e.g., by performing areceptor binding or gene reporter assay concurrently with a cellviability/apoptosis assay).

Despite the undoubted benefits of flow cytometry in drug discovery andother applications, the throughput of modern flow cytometry (e.g., onthe order of 10,000 cells per second) is insufficient to performscreening of the large compound libraries available today atpharmaceutical and biotechnology companies. For high throughputscreening to yield a reasonable number of hits in a short period oftime, hundreds of thousands of compounds must be screened each day.Further, the sheer cost of developing and performing screening assaysdemands that they are completed in a reasonable amount of time, giventhe already long and expensive further testing and clinical trials thatawait such candidate drugs.

Efforts have been made to improve the speed of cytometry and samplehandling, such as development of the HyperCyt® autosampler fromIntellicyt Corporation®. These efforts have enabled the use of flowcytometry at improved speeds, enabling a 384-well plate to be screenedusing 1-microliter samples in approximately 20 minutes. This advance hasopened the door for using flow cytometry in drug screening, yet thetechnique is still at least an order of magnitude slower thanfluorescence plate or microarray readers.

While the HyperCyt autosampler has vastly improved the ability of flowcytometry to perform compound screening, the instrument seriallymultiplexes samples from plate wells into a single stream. It caninterrogate only one well at a time, which limits the throughput (inwells/hour) of the entire screening system. Conventional flow cytometersoffer sufficient throughput (˜10,000 cells/second) to interrogate all ofthe cells in the microliter volumes sampled by the HyperCyt during eachwell period, and as such, the autosampler is the bottleneck to the speedof the screen. At 20 minutes per 384 well plate, this system is capableof examining approximately 25,000 wells per day. While this represents asubstantial throughput, it is four times slower than the industrystandard HTS goal of 100,000 wells (or more) per day. While there areongoing efforts to improve on this number by running several flowcytometers in parallel from multi-probe autosamplers, the cost of thesemulti-instrument systems quickly becomes prohibitively large.Additionally, the complexity of calibrating and controlling multipleindependent instruments can yield unreliable data, making interpretationof assay data difficult.

Regardless of these difficulties, the Genomics Institute of the NovartisResearch Foundation (GNF) has recently built a multimillion dollarhigh-throughput flow cytometry screening system, which at its core,consists of a three-probe autosampler attached to three independentBeckman-Coulter CyAn flow cytometers. Due to the rich assay data itprovides, GNF personnel have reportedly used this system intensely. Thisexample clearly demonstrates the utility of flow cytometry in drugdiscovery, and the need to improve the cost and simplicity of thesesystems.

Accordingly, a need exists for a parallel flow cytometry apparatus andmethod that can significantly increase throughput. The presentdisclosure provides this increased throughput while overcomingshortcomings of previous approaches.

BRIEF SUMMARY

The disclosed parallel flow cytometry overcomes numerous limitationsfound in current instrumentation by employing simultaneous probing ofparallel flow channels in a new manner. By way of example, and not oflimitation, the disclosure combines photomultiplier tube (PMT) basedfluorescence analysis using radio-frequency-tagged emission (FIRE)optical detection scheme with a microfluidic device.

An optical engine is disclosed for a highly-parallel flow cytometerwhich improves sample screening throughput by an order of magnitude,such as 200 wells per minute. In at least one embodiment, the disclosedapparatus and method can be integrated with a commercial multi-probeautosampler.

The disclosed technology drastically reduces both the time and costrequirements of high throughput compound screens using flow cytometry.Inherently, the ability to use multi-parameter flow cytometry providesricher information than instruments, such as plate readers, due to thehigh content cell-level information provided, and the ability to measuresub-populations within the sample wells. While high content imaging alsoprovides many of these advantages, it is not very effective whenutilized with suspension cells, and requires sophisticated imageprocessing and data handling to extract meaningful parameters fromsamples.

Further aspects of the presented technology will be brought out in thefollowing portions of the specification, wherein the detaileddescription is for the purpose of fully disclosing preferred embodimentsof the technology without placing limitations thereon.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The disclosed technology will be more fully understood by reference tothe following drawings which are for illustrative purposes only:

FIG. 1A through FIG. 1D are block diagrams of parallel flow cytometryusing radio frequency tagging of parallel channels using a singleoptical system according to an embodiment of the present disclosure.

FIG. 2A through FIG. 2G are images captured using high-speed imagingflow cytometry according to an embodiment of the present disclosure.

FIG. 3 is an image of parallel flow cytometry such as performedaccording to embodiments of the present disclosure.

FIG. 4A and FIG. 4B are scatter plots of data collected at one or morediscrete sites on a particle such as performed according to embodimentsof the present disclosure.

DETAILED DESCRIPTION

1. Innovation.

The present disclosure provides a single cytometer that is capable ofsampling and reading a plurality of wells (e.g., 10) in parallel, forenhancing flow cytometry throughput, such as utilized in applicationslike drug discovery. Although many commercial options for parallelliquid sample sipping/aspiration and handling, there have been noexisting instruments that could interrogate multiple cells in parallelat high speed with sufficient optical sensitivity. While severalmicrofluidic approaches to parallel flow cytometry have beendemonstrated, these systems employed laser scanning illumination, orsilicon high-speed cameras as optical detectors. These demonstrationshad two primary limitations: (1) laser scanning does not supportparallel analysis of cells flowing at meter/s velocities due to thelimited duty cycle of the laser exposure on each channel; (2) siliconcameras although useful for imaging brightly-illuminated scenes at highspeed, neither possess (i) sufficient shutter speed to avoid image blur,(ii) sufficient readout speed for the high event rates in flowcytometry, nor (iii) the required shot-noise limited optical sensitivityto accurately detect the small number of fluorescence photons emitted bycells during their microsecond transit times through the opticalinterrogation regions. The system of the present disclosure does notsuffer from these shortcomings.

A flow cytometry system of one embodiment, is configured forinterrogating a plurality (e.g., from two to ten or more) independentfocused streams of cells with multi-color fluorescence (FITC, PE), andforward and side-scatter detection. In this embodiment, a FIRE opticalengine is preferably utilized in combination with an inertially focusedmicrofluidic chip. It should be appreciated, however, that the presentdisclosure may be implemented with cells that are focused usinghydrodynamic focusing, sheath flow focusing, acoustic focusing, othertypes of particle focusing methods, and combinations thereof. Inaddition, it should be appreciated that although described as beingutilized with cells, the device of the present disclosure can beutilized for analyzing streams of various particles, including cells,beads, pieces of cells, and so forth.

Another embodiment accomplishes the forgoing by further includingfrequency tagging of the excitation/emission light incident on theplurality of separate flow microchannels allowing detection and analysisin a low-cost single optical system. This obviates the need for manyparallel optical trains, filters, and expensive detectors. As aconsequence of every flow channel being illuminated at a differentmodulation frequency, a single PMT detector can be utilized to detectlight (of a single color, as a fluorescence filter is used) frommultiple points, when the resulting electrical signal is analyzed usingsignal processing. Signals from particles in each flow channel are thusencoded in the frequency domain.

By utilizing the disclosed technology, each stream in the system becomescapable of measurement throughput comparable to modern flow cytometers,(e.g., greater than 10,000 events/second), while the overall system willbe capable of simultaneously handling a plurality, in this example 10,independent samples, thereby speeding up HTS using flow cytometry by anorder of magnitude. Using a different microfluidic chip, the disclosedtechnology will also be adaptable to handle single samples at ratesexceeding 100,000 events/second for other applications, such as rarecell detection (circulating tumor cells, cancer stem cells, circulatingendothelial cells, and so forth.), or simply to speed up dataacquisition from large samples.

The benefits of the combination of these innovations in such a systemcan be summarized as follows: (a) FIRE is configured on the system toindependently-control the illumination of each parallel flow stream,which is critical to the system calibration in order to establish eachflow channel with identical optical sensitivity. (b) FIRE is configuredon the system to utilize a single PMT for each fluorescence or scattermeasurement, avoiding the use of slow and insensitive cameras. Thismeans the number of PMT's does not scale linearly with the samplethroughput of the system, but rather with the number of parametersmeasured. Each PMT has a fluorescence emission filter in front of it,such that it detects one color of fluorescence emission from all flowchannels at the same time. The operational principles of the technologyare described in greater detail below.

A. Fluorescence Imaging Using Radiofrequency-Tagged Emission (FIRE)

FIRE is an ultra-high speed fluorescence imaging technique developed atUCLA to meet the speed demands of high throughput fluorescence imagingflow cytometry and sub-millisecond fluorescence microscopy. The centralfeature of FIRE microscopy is its ability to excite fluorescence in eachindividual point of the sample at a distinct radiofrequency, whichallows detection of fluorescence from multiple points using a singlephotodetector. This excitation occurs at the beat frequency between twointerfering, frequency-shifted beams of light.

FIG. 1A through FIG. 1D illustrate one embodiment 10 of utilizing FIREfor parallel flow cytometry using radiofrequency multiplexing accordingto the present disclosure. A laser 12 (e.g., 488-nm excitation) outputsbeam 14, which is split by beamsplitter 16 (e.g., non-polarizing) intotwo beams 18 a, 18 b in the arms of a Mach-Zehnder interferometer. Thelight in the first arm 18 a is frequency shifted by an acousto-opticdeflector (AOD) 22, which is driven by a phase-engineered radiofrequencycomb 24, designed to minimize signal peak-to-average power ratio. Asseen in FIG. 1B, this RF comb causes AOD 22 to generate multipledeflected optical beams 24 possessing a range of both output angles aswell as frequency shifts. Output from AOD 24 is returned back forcombination with the other arm by mirror 26. It should be appreciatedthat interoperation between the excitation beam with a plurality offluid channels requires the RF comb generator to be configured togenerate a spatially disparate amplitude modulated beam. If configuredfor collecting images, the frequencies in the comb are too closelyspaced for analyzing separate flow streams. This configuration of the RFcomb generator could be referred to as a ‘sparse’ frequency mode.

The second arm of the interferometer has beam 18 b reflected from mirror20 to then be shifted in frequency using an acousto-optic frequencyshifter (AOFS) 28 receiving a signal from a direct digital synthesis(DDS) radio-frequency (RF) tone generator 30 to generate a localoscillator (LO) beam 32.

A cylindrical lens 33 placed after the AOFS matches the angulardivergence of the LO arm to that of the RF comb beams. After combiningbeams 27 and 32 at a second beamsplitter 34, the two beams are focused36 coincident to a line on the sample using a conventional laserscanning microscope lens system. Other configurations of one or moreacousto-optic devices to generate frequency-shifted coincident pairs ofbeams are also disclosed. Alternatively, other system configurations,such as multiple electro-optic, liquid crystal, or other lightmodulators can be employed to amplitude modulate multiple independentexcitation optical beams at more than one unique frequency, such that asingle PMT detector can be used to analyze the fluorescence or scatteremission from all flow channels simultaneously.

The microscope lens system is shown with a dichroic mirror (DM) 38 thatprovides different reflective/transmissive properties at two differentwavelengths. Dichroic mirror 38 reflects the pump laser light, andtransmits longer wavelength light, while dichroic mirror 50 splits thedifferent colors of fluorescence emission so that each PMT can analyzethe amount of light in the different spectral bands. It will beappreciated that dichroic mirror 50 operates in combination with filters46, 48, as a means for separating different colors of fluorescenceemission. The present invention is not limited to using a mirror-filtercombination for separating bands of fluorescence emission, as a numberof techniques are known for performing optical separation with respectto frequency band. Beam 54 from DM 38 is reflected from mirror 56through a lens system 58, 60 to an objective 62, in which the flowchannels 64 are coupled for being read. It should be recognized that theoptics in the illustrated configuration are not configured with angularscanning mechanisms, such as using a scanning mirror for mirror 56, asmight be utilized when capturing images. In the present disclosure, datais collected on a discrete point (or a few points) across the width of asample from which analysis is performed.

An optical signal returned back from objective 62 passes through thelenses 58, 60, mirror 56 and through DM 38 as beam 52, strikes thesecond DM 50 and separates into beams that pass through fluorescenceemission filters (EF) 46, 48, to photomultiplier tubes 42, 44, which arebeing read by a digitizing storage system 40, such as an analog todigital converter, a digitizing oscilloscope, a multi-channel lock-inamplifier, or another high speed data acquisition system.

It should be appreciated that the photomultiplier tubes are not imagingdevices in their conventional use, but operate to multiply photonactivity from a source beam, and convert it to an electrical output.Each PMT is utilized for collecting information about a specificcharacteristic of the particles being analyzed, because these differentcharacteristics have been tagged with fluorophores operating atdifferent wavelengths. It is recognized that fluorophores absorb lightenergy of a specific wavelength (range) and re-emit light at a longerwavelength.

In FIG. 1C, the optical wave interaction at beamsplitter 34 is depictedwith a first signal 27 (ω₀+ω_(LO)) interacting with second signal 32Σ(ω₀+ω_(RF) _(N) ) to output a combined signal 36 Σ(ω_(RF) _(N)−ω_(LO)). These output beams represent the range of frequenciescontained in the amplitude modulated beams that excite particles in theparallel flow channels 70, shown in FIG. 1D.

Since fluorescent molecules in the sample function as square-lawdetectors (their excitation responds to the square of the total electricfield), the resulting fluorescence is emitted at the various beatsdefined by the different frequencies of the two arms of theinterferometer. Further, since acousto-optic devices are inherentlyresonant devices, the acousto-optic frequency shifter (AOFS) 28 in thesecond arm of the interferometer is chosen to heterodyne the beatfrequencies to baseband, in order to maximize the useable bandwidth fora given fluorophore. In this case, an AOFS is utilized, but otherimplementations utilize a second AOD or other acousto-optic device, andcan be driven by a single electronic tone, or a frequency comb.

For the sake of simplicity of illustration, FIG. 1A depicts only twoPMTs being used, however, more PMTs can be added for simultaneouslyregistering additional particle characteristics across the plurality offlow channels. In one embodiment, cylindrical lens 33 placed after theAOFS to match the divergence of the LO beam to that of the RF beams, isreplaced by using two AODs (at the position of the AOFS, but with nocylindrical lens used) of opposite diffraction orders are used togenerate discrete interrogation beams at the sample.

FIG. 1D is a close-up view of an example embodiment 62 of themulti-channel interrogation approach of the present disclosure. Cells inmultiple flow channels 70 (w₁, w₂, w₃, . . . w_(N)) are excited inparallel by beams 68 modulated at unique beat frequencies, passingthrough objective 64 and lens 66. In at least one embodiment (notshown), forward scatter PMT detector is included in the final cytometerdesign. Backscatter detection can replace the conventional side-scatterdetection channel, by using a PMT in a similar position to PMTs 42 and44.

FIRE operates by simultaneously exciting fluorescence from distinctpoints on the sample at a unique radiofrequency. Since the excitation(and hence, emission) from each point is tagged with a unique frequency,a single PMT can be used to collect epi-fluorescence from multiplespatial points, and a Fourier transform of the output signal is used toanalyze the fluorescence emission of the sample, for example, of anarray of parallel flow channels. To excite fluorescence at pre-definedlocations in multiple individual streams of cells, the optical design isconfigured such that a plurality of discrete points (e.g., 10 discretepoints) is illuminated by amplitude modulated beams or pairs offrequency-shifted beams. Illumination at the plurality of discretepoints is a configuration to maximize the amount of laser power incidentupon each flow channel, without wasting laser power on the regionsbetween the flow channels, as would be the case when using an AOFS andcylindrical lens, although this embodiment of the system still enablesanalysis of multiple flow channels using single element detectors.

FIG. 2A through FIG. 2G depict results from experiments using FIRE toperform imaging flow cytometry to show what fluorescence imaging candepict from a single particle in a stream. In this example, MCF-7 breastcarcinoma cells are shown flowing in a microfluidic channel at avelocity of 1 m/s. All images are of MCF-7 breast carcinoma cells,stained with the DNA stain Syto16, taken using a 60×, 0.70-NA objectivelens. In FIG. 2A is seen representative FIRE images of cells flowing ata velocity of 1 m/s, taken using a pixel frequency spacing of 800 kHz,and 54 μW of 488-nm laser power per pixel, measured before theobjective. FIG. 2B and FIG. 2C depict single 10-μs as exposure frametransfer EMCCD images of individual cells flowing at a velocity of 1 m/sfor comparison. The electron multiplier gain was adjusted to producemaximal image SNR, and the EMCCD vertical shift time was set to theminimum value of 564 ns. Blur is observed in the image due to the longexposure time and the frame transfer nature of the EMCCD. In FIG. 2Dthrough FIG. 2G wide field fluorescence images are represented ofstationary MCF-7 cells for morphological reference. All scale bars are10 μm. However, it should be appreciated that the present disclosure isnot configured for performing imaging on cells in a single stream, butinstead to simultaneously analyzing at least one discrete point in eachof a plurality of flow streams. Examples of the types of informationprovided by the presented technology are described in FIG. 3 throughFIG. 4B.

These preceding tests serve to illustrate the ability of the FIREtechnology to use a single PMT detector to simultaneously interrogatecellular fluorescence with high sensitivity (54 μW of laser excitationpower is used per pixel) from 125 distinct spatial pixels at high speed.By reducing the number of pixels from 125 to 10 (using one ˜50 μm×10 μm“pixel” per flow stream) the optical sensitivity of the system improvesdramatically (more than 10 dB). This is due to the fact that (i) thenumber of pixels multiplexed into the same PMT is reduced and (ii) shotnoise crosstalk that results in multiple pixels being “on” at the sametime is dramatically reduced, as the cell arrival times at the opticalinterrogation region on the microfluidic chip follows Poissonstatistics, as opposed to the case of imaging, in which multipleadjacent pixels in an image are typically “on” simultaneously. However,if multiple cells are interrogated at the same time, digitalpost-processing can perform compensation of the fluorescence intensitiesto account for this shot noise crosstalk. The concept of compensation isubiquitous in flow cytometry, and is typically used to account for thebroad emission spectra of fluorophores. Shot noise compensation isactually simpler than spectral compensation, in that the compensationvalues are simply proportional to the square root of the average outputcurrent of the detector.

2. Methods and Materials

Design and Implementation of Parallel Optical Cytometry Engine andParallel Microfluidic Chip.

The field of view, cell flow velocities, inertial focusing spatialdistribution, and excitation laser spot sizes are designed for producinga combination of maximum throughput as well as maximum signal-to-noiseratio (SNR). The design goal is to record 10,000 events/second in eachof a plurality of parallel channels (e.g., 10), all within a 1 mm fieldof view. By way of example and not limitation, a 488 nm laser isutilized for excitation, and existing digitizing electronics (e.g.,16-bit with memory depth capable of continuously storing data from morethan 10″9 cells) are utilized to collect the data. Higher bit-depthcommercially-available digitizers can be used for better intensityresolution, as desired. As the optical excitation and collectionefficiency will vary from channel to channel, variations in the signalsare measured using fluorescent reference beads. To eliminate thisvariation, the laser power directed to each channel is adjusted, such asin software, by adjusting the MATLAB-generated waveform used to drivethe acousto-optics. A 0.8-NA (or higher numerical aperture), 20×microscope objective is utilized to improve the detection sensitivity ofthe system (as compared to 0.45-NA, 20×). This parameter can be measuredwith the system of the present disclosure using standard techniques, andlaser power can be increased to achieve this goal if not possible with a100 mW 488 nm laser. More powerful lasers (e.g., greater than 1 W) existthat would further improve the sensitivity (dividing the power into 10spots reduces the power per channel to approximately 50 mW per channel).

A. FIRE with Parallel Flow Channels.

From previous testing templates have been evaluated for fabricatingmassively parallel microfluidic chips. The present disclosure can beutilized separately with these chips, which have walls in between eachstream. A variety of microfluidic chip designs can be envisioned thatwill work with the frequency multiplexed nature of the FIRE parallelflow cytometer. It should also be appreciated that hydrodynamicfocusing, inertial focusing, or other techniques and combinationsthereof can be utilized in the flow channels to align the cells in avariety of positions for interrogation using modulated optical beams.

FIG. 3 depicts a parallel flow through microfluidic channels, in whichparticles in those channels are all simultaneously detected/capturedaccording to the presented technology. In the figure one sees aplurality of parallel flow channels, each marked with the vertical flowarrow. By way of example the image was captured with only two particlesin the channels. In this demonstration, excitation and fluorescentresponse capture was performed using a similar setup to that of thedisclosed technology. To demonstrate the ability of FIRE to perform flowcytometry measurements on distinct flow channels, single-point flowcytometry measurements were performed on fluorescent beads. The FIREsetup shown in the figure excites two parallel microfluidic channels ona chip fabricated using polydimethylsiloxane (PDMS) molding. A singlesample, consisting of 1.0 μm (λ_(ex)/λ_(em) 515/585 nm) and 2.2 μm(λ_(ex)/λ_(em) 490/520 nm) fluorescent beads, was flowed throughparallel channels at a mean velocity of 1 m/s using a syringe pump.Channels 1 and 2 were excited using a 488 nm laser, modulated at beatfrequencies of 10 and 50 MHz, respectively.

FIG. 4A and FIG. 4B depict scatter plots for the 10 MHz and 50 MHz flowchannels after raw data was thresholded and the fluorescence pulses wereintegrated to create PE (red) vs. FITC (green) fluorescence intensityscatter plot. It will be noted that these plots are shown asmonochromatic to simplify reproduction of the patent application. Thetwo bead populations in both channels are clearly resolved, despite thelow (8-bit) resolution of the digitizer used to collect the data (no logamplification was used). This experiment was run at a total throughputof approximately 70,000 beads/second (35,000 beads/second/flow channel).This preliminary result clearly shows the ability of FIRE to perform2-color parallel conventional flow cytometry using a singlephotomultiplier tube detector to detect each fluorescence colorsimultaneously from multiple flow channels.

From the description herein, it will be appreciated that the presentdisclosure encompasses multiple embodiments which include, but are notlimited to, the following:

1. An apparatus for simultaneously analyzing physical and chemicalcharacteristics of particles in multiple streams of particles, theapparatus comprising: (a) at least one optical excitation source; (b) afirst radio-frequency source having a first radio-frequency output; (c)a second radio-frequency source having a second radio-frequency output;(d) an optical, or acousto-optical, combining device configured forcombining said first radio-frequency output and said secondradio-frequency output into an optical interrogation beam having aspatially distributed beat frequency, wherein said optical interrogationbeam comprises a plurality of separate beams configured for beingdirected to each of a plurality of focused streams; (e) an opticalsystem configured for directing said optical interrogation beam acrossthe plurality of focused streams of particles so that said spatiallydistributed beat frequency simultaneously spans said plurality offocused streams of particles whose physical and chemical characteristicsare being analyzed by said apparatus; and (f) an optical detectorconfigured for registering fluorescence of particles, within saidplurality of focused streams of particles, at different modulationfrequencies within said spatially distributed beat frequency, whereinfluorescence across multiple focused streams of particles are registeredin parallel by said optical detector.

2. The apparatus of any preceding embodiment, wherein said opticalexcitation source comprises a laser.

3. The apparatus of any preceding embodiment, wherein said lasercomprises a continuous wave laser.

4. The apparatus of any preceding embodiment, wherein said opticalcombining device, said optical system, or a combination thereof, areconfigured for independently-controlling illumination directed at eachparallel flow stream, toward establishing each flow channel withidentical optical sensitivity.

5. The apparatus of any preceding embodiment, wherein said plurality offocused streams of particles are retained in a microfluidic device orchip.

6. The apparatus of any preceding embodiment, further comprising one ormore additional optical detectors, each of which are configured forregistering fluorescence at a different modulation frequency so thatdifferent characteristics of particles in each focused stream aredetected.

7. The apparatus of any preceding embodiment, further comprising opticalmeans for separating different colors of fluorescence emission so thateach of these multiple detectors can analyze fluorescence in a differentspectral band associated with different characteristics of particles ineach focused stream of particles.

8. The apparatus of any preceding embodiment, wherein each channel of amicrofluidic device or chip is exposed to excitation at a differentmodulation frequency that is unique to that channel by using beatfrequency modulation.

9. The apparatus of any preceding embodiment, wherein fluorescence isexcited in each channel at a distinct radio-frequency, which allowsdetection of fluorescence from multiple points using a singlephotodetector.

10. The apparatus of any preceding embodiment, wherein said particlescomprise cells, or portions of cells.

11. The apparatus of any preceding embodiment, wherein said optical, oracousto-optical, combining device comprises an interferometer, with thebeam being split with a first arm of the beam received at an firstacousto-optic device, and a second arm of the beam received by a secondacousto-optic device, after which the arms of the beam are recombinedand directed through the optical system.

12. The apparatus of any preceding embodiment, wherein said first orsecond acousto-optic device comprises an acousto-optic deflector (AOD),or an acousto-optic frequency shifter (AOFS).

13. The apparatus of any preceding embodiment, further comprising aradio frequency (RF) comb generator configured for driving saidacousto-optic deflector thus generating a set of spatially disparateamplitude modulated beams through beat frequency modulation withsufficient spatial width to span said plurality of focused streams ofparticles.

14. The apparatus of any preceding embodiment, further comprisingdigitizing electronics configured for storing data on registeredfluorescence of particles to allow for analysis of particlecharacteristics.

15. An apparatus for simultaneous analyzing physical and chemicalcharacteristics of particles in multiple streams of particles, theapparatus comprising: (a) at least one optical excitation source; (b) afirst radio-frequency source having a first radio-frequency output; (c)a second radio-frequency source having a second radio-frequency output;(d) an optical, or acousto-optical, combining device including aninterferometer with the optical excitation source split into a first armreceived at a first acousto-optic device, and a second arm received by asecond acousto-optic device, after which the arms of the beam arerecombined; (e) wherein said first or second acousto-optic devicecomprises an acousto-optic deflector (AOD), or an acousto-opticfrequency shifter (AOFS); (f) a radio frequency (RF) comb generatorconfigured for driving said acousto-optic deflector (AOD) to generate aset of spatially disparate amplitude modulated beams through beatfrequency modulation with sufficient spatial width to span saidplurality of focused streams of particles; (g) wherein an opticalinterrogation beam having a spatially distributed beat frequency isoutput from said optical, or acousto-optical, combining device; (h) anoptical system configured for directing said optical interrogation beamacross a plurality of focused streams of particles so that saidspatially distributed beat frequency simultaneously spans said pluralityof focused streams of particles whose physical and chemicalcharacteristics are being analyzed by said apparatus; and (i) an opticaldetector configured for registering fluorescence of particles, withinsaid plurality of focused streams of particles, at different modulationfrequencies within said spatially distributed beat frequency, whereinfluorescence across multiple focused streams of particles are registeredin parallel by said optical detector whose output is configured forreceipt by digitizing electronics configured for storing data onregistered fluorescence of particles to allow for analysis of particlecharacteristics.

16. The apparatus of any preceding embodiment, wherein said opticalexcitation source comprises a laser.

17. The apparatus of any preceding embodiment, wherein said lasercomprises a continuous wave laser.

18. The apparatus of any preceding embodiment, wherein said opticalcombining device, said optical system, or a combination thereof, areconfigured for independently-controlling illumination directed at eachparallel flow stream, toward establishing each flow channel withidentical optical sensitivity.

19. The apparatus of any preceding embodiment, wherein said plurality offocused streams of particles are retained in a microfluidic device orchip.

20. The apparatus of any preceding embodiment, further comprising one ormore additional optical detectors, which are each configured forregistering fluorescence at a different modulation frequency so thatdifferent characteristics of particles in each focused stream aredetected.

21. The apparatus of any preceding embodiment, further comprising anoptical device, or devices, for separating different colors offluorescence emission so that each of these multiple detectors cananalyze fluorescence in a different spectral band associated withdifferent characteristics of particles in each focused stream ofparticles.

22. The apparatus of any preceding embodiment, wherein each channel of amicrofluidic device or chip is exposed to excitation at a differentmodulation frequency that is unique to that channel by using beatfrequency modulation.

23. The apparatus of any preceding embodiment, wherein fluorescence isexcited in each channel at a distinct radio-frequency, which allowsdetection of fluorescence from multiple points using a singlephotodetector.

24. The apparatus of any preceding embodiment, wherein said particlescomprise cells, or portions of cells.

25. The apparatus of any preceding embodiment, further comprisingdigitizing electronics configured for continuously storing data onregistered fluorescence of particles to allow for analysis of particlecharacteristics.

26. A method for performing flow cytometry in simultaneouslyinterrogating physical and chemical characteristics of particles inmultiple streams of particles, the method comprising: (a) introducingfluidic particles, or cells, as targets of interest into a plurality offluid flow channels; (b) simultaneously exposing the targets to anexcitation source as they flow though the fluid flow channels, so thattargets in each fluid flow channel are exposed to a different modulatedoptical beam with a modulation frequency that is unique to that channel;(c) detecting fluorescence from said targets based on excitation at aparticular modulation frequency as detected by an optical detector; and(d) outputting fluorescence data of said targets for analysis ofphysical and chemical characteristics of said targets in a fluid of saidfluid flow channels.

27. A method for performing flow cytometry, the method comprising: (a)providing a microfluidic chip having a plurality of parallel flowchannels; (b) introducing fluidic targets of interest into a pluralityof said parallel flow channels; (c) simultaneously exposing the fluidictargets to an excitation source as they flow though the parallel flowchannels; (d) wherein each said parallel flow channel is exposed to adifferent modulated optical beam with a modulation frequency that isunique to that channel; and (e) using a single detector, detectingfluorescence from the targets based on excitation at a particularmodulation frequency to analyze physical and chemical characteristics ofparticles in a fluid.

28. The method of any preceding embodiment, wherein each channel isexposed to excitation at a different modulation frequency that is uniqueto that channel by using beat frequency modulation.

29. The method of any preceding embodiment, wherein each wherein eachchannel is exposed to excitation at a different modulation frequencythat is unique to that channel by using a separate modulation source foreach channel.

30. The method of any preceding embodiment, wherein fluorescence isexcited in each channel at a distinct radiofrequency, which allowsdetection of fluorescence from multiple points using a singlephotodetector.

31. The method of any preceding embodiment, wherein excitation occurs atthe beat frequency between two interfering, frequency-shifted beams oflight.

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

What is claimed is:
 1. An apparatus comprising: a laser; a frequencyshifter component configured to generate from a beam of light from thelaser a first beam of frequency shifted light and a second beam offrequency shifted light; a flow cell comprising a first flow channel anda second flow channel; and an optical apparatus configured to direct thefirst beam of frequency shifted light onto the first flow channel andthe second beam of frequency shifted light onto the second flow channel.2. The apparatus according to claim 1, wherein the frequency shiftercomponent comprises: a first radio-frequency source having a firstradio-frequency output; a second radio-frequency source having a secondradio-frequency output.
 3. The apparatus according to claim 1, whereinthe frequency shifter component comprises an acousto-optical combinerconfigured to produce an optical interrogation beam comprising the firstbeam of frequency shifted light and the second beam of frequency shiftedlight.
 4. The apparatus according to claim 3, wherein theacousto-optical combiner is configured to produce the opticalinterrogation beam by combining a first radio-frequency output and asecond radio-frequency output with the beam of light from the laser. 5.The apparatus according to claim 3, wherein the acousto-optical combineris configured to independently control the intensity of the first beamof frequency shifted light and the intensity of the second beam offrequency shifted light.
 6. The apparatus according to claim 1, whereinfirst beam of frequency shifted light is spatially distributed from thesecond beam of frequency-shifted light.
 7. The apparatus according toclaim 1, further comprising a photodetector that is configured tosimultaneously detect fluorescence from particles at a first modulationfrequency moving through the first flow channel and fluorescence fromparticles at a second modulation frequency moving through the secondflow channel.
 8. The apparatus according to claim 7, wherein the firstmodulation frequency is different from the second modulation frequency.9. The apparatus according to claim 7, wherein fluorescence detected atthe first modulation frequency and fluorescence detected at the secondmodulation frequency are registered in parallel.
 10. The apparatusaccording to claim 7, wherein the photodetector is configured tosimultaneously detect fluorescence from particles moving at a rate of 1m/s through the first flow channel and the second flow channel.
 11. Theapparatus according to claim 1, wherein the first flow channel and thesecond flow channel have substantially the same optical sensitivity. 12.The apparatus according to claim 1, wherein the frequency shiftercomponent comprises one or more of an acousto-optic deflector (AOD) andan acousto-optic frequency shifter (AOFS).
 13. The apparatus accordingto claim 12, further comprising a radio-frequency comb generatorconfigured to produce the first beam of frequency shifted light and thesecond beam of frequency shifted light, wherein the first beam offrequency shifted light and the second beam of frequency shifted lightare amplitude modulated and spatially disparate.
 14. The apparatusaccording to claim 13, wherein the first beam of frequency shifted lightand the second beam of frequency shifted light have a spatial widthsufficient to span the width of the first flow channel and the width ofthe second flow channel.
 15. The apparatus according to claim 1, whereinthe laser is a continuous wave laser.
 16. The apparatus according toclaim 1, wherein the flow cell is a microfluidic device comprising aplurality of parallel flow channels.
 17. The apparatus according toclaim 16, wherein the frequency shifter component is configured togenerate from the beam of light from the laser an interrogation beamcomprising a plurality of spatially distributed beams of frequencyshifted light.
 18. The apparatus according to claim 17, wherein theoptical apparatus is configured to direct each of the plurality of beamsof frequency shifted light onto the plurality of parallel flow channels.19. The apparatus according to claim 1, further comprising aphotodetector.
 20. The apparatus according to claim 19, wherein thephotodetector is a single photomultiplier tube (PMT).